fpls-09-00443 April 4, 2018 Time: 16:15 # 1

REVIEWpublished: 06 April 2018

doi: 10.3389/fpls.2018.00443

Edited by:Alexander Arthur Theodore Johnson,

University of Melbourne, Australia

Reviewed by:Francesco Di Gioia,

University of Florida, United StatesAymeric Goyer,

Oregon State University,United States

*Correspondence:Dominique Van Der Straeten

Dominique.VanDerStraeten@ugent.be

Specialty section:This article was submitted to

Plant Nutrition,a section of the journal

Frontiers in Plant Science

Received: 19 January 2018Accepted: 21 March 2018

Published: 06 April 2018

Citation:Strobbe S and Van Der Straeten D

(2018) Toward Eradicationof B-Vitamin Deficiencies:

Considerations for Crop Biofortification.Front. Plant Sci. 9:443.

doi: 10.3389/fpls.2018.00443

Toward Eradication of B-VitaminDeficiencies: Considerations forCrop BiofortificationSimon Strobbe and Dominique Van Der Straeten*

Laboratory of Functional Plant Biology, Department of Biology, Ghent University, Ghent, Belgium

‘Hidden hunger’ involves insufficient intake of micronutrients and is estimated to affectover two billion people on a global scale. Malnutrition of vitamins and minerals isknown to cause an alarming number of casualties, even in the developed world. Manystaple crops, although serving as the main dietary component for large populationgroups, deliver inadequate amounts of micronutrients. Biofortification, the augmentationof natural micronutrient levels in crop products through breeding or genetic engineering,is a pivotal tool in the fight against micronutrient malnutrition (MNM). Although theseapproaches have shown to be successful in several species, a more extensiveknowledge of plant metabolism and function of these micronutrients is required to refineand improve biofortification strategies. This review focuses on the relevant B-vitamins(B1, B6, and B9). First, the role of these vitamins in plant physiology is elaborated,as well their biosynthesis. Second, the rationale behind vitamin biofortification isillustrated in view of pathophysiology and epidemiology of the deficiency. Furthermore,advances in biofortification, via metabolic engineering or breeding, are presented. Finally,considerations on B-vitamin multi-biofortified crops are raised, comprising the possibleinterplay of these vitamins in planta.

Keywords: micronutrients, biofortification, metabolic engineering, folate, pyridoxine, thiamine, cropimprovement, plant development

INTRODUCTION

In an era of tremendous technological capabilities, insufficient accessibility to nutritious food,a primary human need, still affects over two billion people on a global scale (Bailey et al.,2015; Gupta, 2017). Though approximately 800 million people endure energy deficit due toinadequate amounts of calories in their diet (Haddad et al., 2016; FAOSTAT, 2017), the relativeabundance of undernourishment has dropped to almost half in the last 25 years. Unfortunately,the degree of undernourishment witnessed over the last decades has recently passed a minimum,as undernourishment is estimated to have affected 815 million people in 2016, as opposed to 777million in 2015 (FAOSTAT, 2017). The general trend of a decrease in global undernourishment canbe mainly attributed to yield improvement of important staple crops such as rice, maize and wheat,which has more than doubled since 1960 (Long et al., 2015). Unfortunately, caloric malnutritionrepresents only a portion of the food-related burden of diseases, as micronutrient malnutrition(MNM) is present in over one–fourth of the world’s population (Bailey et al., 2015; Blancquaertet al., 2017; De Lepeleire et al., 2017).

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Micronutrient malnutrition comprises shortage of dietarymicronutrients, including minerals (iron, zinc, iodine, selenium,etc.) as well as vitamins (Bailey et al., 2015). Micronutrientmalnourishment, commonly referred to as the “hidden hunger,”is known to induce diseases and disorders in many populations,not particularly confined to the developing world. Pregnantwomen and young children are most vulnerable for MNM, oftenresulting in death (Bailey et al., 2015; De Steur et al., 2015;Win, 2016; De Lepeleire et al., 2017). MNM can be consideredan urgent global concern, persistent in many populations andremaining largely hidden (Ruel-Bergeron et al., 2015). Anemia,a condition of suboptimal hemoglobin level (Scott et al., 2014),illustrates the disastrous impact of combined micronutrientshortage on human physiology, as its occurrence has beenlinked with deficiency in iron (Camaschella, 2015), pro-vitaminA (Semba et al., 1992; West et al., 2007), thiamin (vitaminB1) (Franzese et al., 2017), pyridoxine (vitamin B6) (Clayton,2006; Hisano et al., 2010) and folate (vitamin B9) (Moll andDavis, 2017). Anemia is held responsible for almost 2 milliondeaths of children under 5 years old on a yearly basis (Scottet al., 2014), and is estimated to affect more than 2 billionpeople globally. It is estimated that half of the cases of anemiacould be attributed to deficiency in one or more micronutrients,though primary factors are iron and folate shortage (Scott et al.,2014).

High incidence of micronutrient deficiencies are, in manycases, related to monotonous diets, largely consisting of energyrich, starchy staples (Blancquaert et al., 2017; De Lepeleire et al.,2017). These crops including wheat, rice, potato, cassava, cornand plantain have the tendency to contain inadequate levelsof vitamins and therefore expose the population, consumingmassive amounts of these staples, to the risk of vitamindeficiencies (Table 1). This is a downside of the cheap supply ofenergy rich staples, which enabled the aforementioned decreasein caloric malnourishment.

Given the observation that MNM have a detrimental effecton global human health, there is a great need to stronglyreduce these deficiencies, also stated in the Copenhagenconsensus, where micronutrient interventions were ranked asthe number one priority, related to Sustainable DevelopmentGoal 2 (SDG2), requiring great global investment (CopenhagenConsensus, 2012). Fortunately, there are several means tocombat MNM in an effective way, which can be divided ineducation, supplementation and biofortification. Behavioralinterventions, consisting of educational efforts encouragingdietary diversification, are the ideal means to improve themicronutrient status of a population (Reinbott et al., 2016).This strategy, however, requires changes in cultural orreligious habits of certain communities, as well as recurrentinterventions (Blancquaert et al., 2017). Fortification includesthe administration of micronutrient to the population under theform of pills or fortification of food products (such as flour).The latter method, which is mandatory in many countries, hasproven to be a rapid medium to ensure optimal micronutrientlevels in the troubled populations (Pasricha et al., 2014; Sandjajaet al., 2015; Atta et al., 2016; Rautiainen et al., 2016; Wang et al.,2016). Unfortunately, supplementation depends on specialized

infrastructure and appears difficult to implement in poor ruralpopulations who have the highest demand for micronutrientinterventions (Blancquaert et al., 2017). Luckily, biofortification,which involves the augmentation of the natural nutritional valueof crops, can be addressed as a valuable additional strategy inthe battle against MNM (Blancquaert et al., 2017; Garcia-Casalet al., 2017; Martin and Li, 2017; Strobbe and Van Der Straeten,2017). Biofortification of locally consumed crops does notrequire changes in consumer behavior and demands only aone-time investment (De Steur et al., 2015, 2017; Bouis andSaltzman, 2017). Biofortification of staple crops, massivelyconsumed in deficient populations, is an excellent way tosupply sufficient micronutrients (Blancquaert et al., 2017; DeLepeleire et al., 2017). Biofortification sensu stricto, therebyomitting agricultural interventions (Cakmak and Kutman,2017; Watanabe et al., 2017), comprises breeding techniquesas well as genetic engineering approaches (Blancquaert et al.,2017; Bouis and Saltzman, 2017). Breeding strategies have theadvantage to be easily implemented in agriculture, as theydo not require exhaustive regulations (Mejia et al., 2017).However, the scope of the breeding approaches is confinedto sexual compatibility, thereby lacking the ability to exploituseful animal or prokaryotic derived characteristics (Strobbeand Van Der Straeten, 2017). Biofortification via metabolicengineering, overrules this restriction. Furthermore, the latterapproaches enable creation of a biofortification strategy blue-print, applicable to a wide variety of food crops (Strobbeand Van Der Straeten, 2017). Metabolic engineering does,however, require a great knowledge of the specific micronutrientmetabolism and its importance in the physiology of theplant.

This review reflects upon the acquired knowledge whichenabled successful B-vitamin biofortification in food crops,bundling information on thiamin (B1), pyridoxine (B6),and folates (B9). This evaluation includes vitamin functionin plant growth and development as well as importance inhuman pathophysiology, epidemiology and accomplishments inbiofortification. Furthermore, in view of multi-biofortification,the simultaneous biofortification of multiple vitamins andminerals, possible synergistic or adverse effects of micronutrientcombinations, are scrutinized. Multi-biofortification endeavorsare the step-stone for future eradication of MNM. A list ofabbreviations can be found in Supplementary Table S1.

VITAMIN B1 – THIAMIN

Thiamin is a water soluble B-vitamin consisting of a pyrimidinering, linked to a thiazole moiety by a methylene bridge (Lonsdale,2006) (Figure 1). Vitamin B1 consists of different itamer formsof thiamin, predominantly occurring as thiamin and its differentphosphate esters thiamin pyrophosphate (TPP) and thiaminmonophosphate (TMP). However, other forms of thiamin doexist, such as thiamin triphosphate, though their contributionto the total pool is rather marginal (Gangolf et al., 2010). Inliterature, thiamin(e) is sometimes confusingly used to describethe total pool of the different B1-vitamers, here simply referred to

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TABLE 1 | Vitamin content of six major staple crops.

RDA1 Wheat (Triticumaestivum) Soft,

white

Rice (Oryza sativa)White, long-grain,

regular, raw

Potato (Solanumtuberosum) Flesh

and skin, raw

Cassava(Manihot

esculenta) Raw

Corn (Zeamays) Sweet,

white, raw

Plantain(Musa sp.)

Raw

B1 (mg) 1.4 0.41 (sufficient ) 0.07 (5) 0.081 (4) 0.087 (3) 0.2 (2) 0.052 (8)

B6 (mg) 2 0.378 (sufficient) 0.164 (3) 0.298 (2) 0.088 (4) 0.055 (9) 0.299 (2)

B9 (µg) 600 41 (3) 8 (16) 15 (8) 27 (4) 46 (3) 22 (8)

Highest consumption(g/capita.day)2

/ 609 (Azerbaijan) 4703 (Bangladesh) 502 (Belarus) 678 (DemocraticRepublic of the

Congo)

434 (Lesotho) 350 (Ghana)

Vitamin content data were retrieved from the USDA database (USDA, 2016). RDA data were retrieved from (Trumbo et al., 2001). Values represent vitamin content of100 g fresh edible portion of each crop. Fold enhancement needed to reach the RDA of the corresponding vitamin upon consuming the highest average national serving,is indicated between brackets. Data on staple crop consumption are derived from FAOSTAT (FAOSTAT, 2017). 1Highest recommended daily allowance (RDA). 2 In 2013.3Milled equivalent.

FIGURE 1 | Thiamin biosynthesis in plants. Synthesis of pyrimidine and thiazole moieties as well as their condensation occurs in plastids. Biosynthesis pathway isshown in blue, enzymes in black. Transport across membranes is proposed to be carrier-mediated (barrels), of which the identified mitochondrial TPP carrier isindicated (red barrel) (Frelin et al., 2012). The chemical structure of thiamin is depicted, of which the free hydroxyl group can be pyrophosphorylated by action ofthiamin pyrophosphokinase (TPK). End product feed-back, performed by TPP on the THIC riboswitch, is depicted in red. Products: Gly, glycine; NAD+, nicotinamideadenine dinucleotide; SAM, S-adenosylmethionine; AIR, 5-aminoimidazole ribonucleotide; HET-P, 4-methyl-5-β-hydroxyethylthiazole phosphate; HMP-P,4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate; HMP-PP, HMP-pyrophosphate; TMP, thiamin monophosphate; TPP, thiamin pyrophosphate. Enzymes:THIC, HMP-P synthase; THI1, HET-P synthase; TH1, HMP-P kinase/TMP pyrophosphorylase; TPK, thiamin pyrophosphokinase; TH2, TMP phosphatase, PALEGREEN1.

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as B1. The bioactive vitamer is TPP, serving as cofactor in multipleenzymatic reactions.

BiosynthesisBiosynthesis of TPP in plants takes place in plastids, cytosoland mitochondria (Figure 1). Although the TMP vitamer issynthesized in the chloroplast, subsequent enzymatic reactionsin mitochondria and cytosol are required to finalize thede novo biosynthesis of TPP, the bioactive vitamer (Mimuraet al., 2016; Goyer, 2017; Hsieh et al., 2017). Formation ofTMP involves the synthesis of the thiazole and pyrimidinemoieties, followed by their condensation, all of which occurin chloroplasts (Goyer, 2017). The thiazole moiety of thethiamin structure is created by the action of 4-methyl-5-β -hydroxyethylthiazole phosphate (HET-P) synthase (THI1),requiring NAD+ and glycine as a substrate and yieldingHET-P (Godoi et al., 2006). In this reaction the THI1enzymes is consumed, as it supplies a sulfide from a cysteineresidue, making THI1 a ‘suicidal’ enzyme. 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate (HMP-P) synthase (THIC)is an iron-sulfur cluster containing enzyme, catalyzing theformation of HMP-P, the pyrimidine intermediate of thiaminbiosynthesis (Raschke et al., 2007; Kong et al., 2008; Goyer, 2017).This reaction requires 5-aminoimidazole ribonucleotide (AIR)(derived from purine metabolism) and S-adenosylmethionine(SAM) as substrates (Chatterjee et al., 2008). This is the keyregulatory step of thiamin biosynthesis, as the promotor isunder control of the circadian clock, and the terminator isfeedback inhibited by the end product TPP (Bocobza et al.,2013). This terminator contains a riboswitch, rarely seen ineukaryotes (Wachter et al., 2007). The riboswitch acts by bindingTPP in its pre-mRNA state, resulting in the formation of anunstable splice variant of the THIC gene, thereby causing alowered THIC activity (Wachter et al., 2007; Bocobza et al.,2013). Next, the bifunctional enzyme harboring HMP-P kinaseand TMP pyrophosphorylase activities (TH1), catalyzes theHMP-P phosphorylation and subsequent condensation of HMP-PP and HET-P to form TMP (Ajjawi et al., 2007b). TMPforms the end product of plastidial thiamin biosynthesis andis further processed by the PALE GREEN1/TH2 enzyme, afterexiting the plastids (Mimura et al., 2016; Hsieh et al., 2017).The subcellular localization of TH2 action, being cytosolic,mitochondrial, or both, has been debated. First, the enzyme wasdiscovered in the cytosolic fraction of Arabidopsis (Ito et al.,2011; Mimura et al., 2016). Later experimental evidence, utilizingtranslational fusions with green fluorescent protein (GFP),confirmed the cytosolic location of the TH2 enzyme, realizedfrom a (preferred) native secondary translational initiation site(Mimura et al., 2016). This secondary translational initiationsite yields a protein which lacks the functional N-terminalmitochondrial targeting peptide of TH2. Therefore, TH2 wasconsidered to be predominantly residing in the cytosol thoughlikely also present in mitochondria. More recently, TH2 wasfound to be almost exclusively localized in the mitochondria ofth2/pale green1 rescued with a GFP-fused TH2, controlled bythe cauliflower mosaic virus (CaMV) 35S promoter (Hsieh et al.,2017). These seemingly contradicting findings can be explained

by the fact that a strong constitutive promoter (35S) precedingthe full coding sequence of TH2, favors the first translationalstart site and incorporates the mitochondrial targeting peptideinto the arising protein (Hsieh et al., 2017). However, thesefindings highlight the ability of sole mitochondrial TH2 activityto complement the th2/pale green1 mutant, indirectly hintingat the existence/necessity of a mitochondrial TMP importeras well as a thiamin exporter. Combining these results, itcan be concluded that TMP dephosphorylation, executed byTH2, likely occurs primarily in the cytosol and to a lesserextent in mitochondria. However, it cannot be excluded thatthis subcellular localization of TH2 action might change indifferent conditions/tissues/species. The reaction mediated byTH2 yields thiamin. In turn, thiamin, is the substrate for thiaminpyrophosphokinase (TPK), producing the active vitamer TPP inthe cytosol (Ajjawi et al., 2007a). TPP subsequently travels to thedifferent subcellular locations via carriers. Two mitochondrialTPP carriers have been partially characterized in plants (Frelinet al., 2012).

Role in Plant PhysiologyVitamin B1, sometimes called the ‘energy vitamin,’ plays acrucial role in plant energy homeostasis, mostly by the roleof TPP as a cofactor (Goyer, 2010). TPP is a cofactor forthree enzymes which are central to the energy metabolism.First, the pyruvate dehydrogenase (PDH) complex, catalyzingpyruvate decarboxylation, yielding acetyl CoA and NADH,necessary for the tricarboxylic acid (TCA or Krebs) cycleand biosynthetic processes, respectively (Bocobza et al., 2013).In addition, in the tricarboxylic acid cycle, α-keto-glutaratedehydrogenase (2-oxoglutarate dehydrogenase E1 component,OGDH) functioning also requires TPP, further accentuating itscritical role in central metabolism (Rapala-Kozik et al., 2012).Third, TPP is an essential cofactor of the transketolase (TK)enzyme, playing a key role in the Calvin cycle as well as thepentose phosphate pathway, which renders pentose sugars as wellas NADPH to the cell (Goyer, 2010).

Hence B1, in its form of TPP, controls a few key steps incentral aerobic energy metabolism. In this regard, TPP has beensuggested to influence the flux through these pathways, as it isrequired in their rate-limiting steps (Bocobza et al., 2013). Indeed,modestly increasing TPP concentration, through introduction ofa non-functional riboswitch in Arabidopsis, induced enlargedactivity of TPP-dependent enzymes (PDH, OGDH, and TK)(Bocobza et al., 2013). Moreover, these plants emitted largeramounts of CO2, suggesting overactive oxidative metabolism.This is further confirmed by the observation of depleted starchreserves at the beginning of the light period in high TPP lines(Bocobza et al., 2013). This presents a clear rationale behind thestrict circadian regulation on B1 biosynthesis. As a consequenceof its influence on central metabolism, metabolite composition,particularly that of amino acids, is severely altered in plants withaberrant B1 composition (Bocobza et al., 2013).

B1 metabolism was shown to have a clear function in enablingplants to cope with biotic as well as abiotic stresses. Thiaminbiosynthesis as well as B1 levels were observed to increase uponapplication of abiotic stresses such as high light, drought, salt

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and oxidative stress, conferring tolerance (Kaya et al., 2015; Yeeet al., 2016). Remarkably, this B1-induced tolerance to oxidativestress was concomitant with decreased production of reactiveoxygen species (ROS) (Tunc-Ozdemir et al., 2009). The exactmolecular basis for this role of B1 in stress adaptation of plantsremains partly unknown. Given the enhanced expression ofTPP-dependent enzymes in plants exposed to drought stress,the influence of B1 on abiotic stress control seems to be viaits end product TPP (Rapala-Kozik et al., 2012). On the otherhand, the B1 biosynthesis enzyme THI1, responsible for plastidialthiazole biosynthesis, appears to be able to directly regulatestomatal closure (Li et al., 2016). The potential of B1 to enhanceabiotic stress tolerance was, however, not observed in engineeredArabidopsis lines (Dong et al., 2015). Considering biotic stresses,B1 has been shown to confer systemic acquired resistance(SAR) (Ahn et al., 2007; Bahuguna et al., 2012; Boubakri et al.,2012). Thiamin-treated plants depicted enhanced ROS (hydrogenperoxide, produced upon up-regulation of superoxide dismutase)accumulation upon infection (Bahuguna et al., 2012), contrastingtheir role of decreasing ROS in abiotic stresses (Tunc-Ozdemiret al., 2009; Kaya et al., 2015). By doing so, thiamin provokespriming, a state in which the plant has the ability to reactmore rapidly upon infection (Conrath et al., 2006; Ahn et al.,2007). This priming effect, described for B1, was confirmed inhigh B1 engineered Arabidopsis (Dong et al., 2015), but notseen in rice (Dong et al., 2016). Moreover, thiamin treatmentof plants induced higher accumulation of phenolic compounds,salicylic acid (SA) (through higher phenylalanine ammonialyase activity) and nitrogen assimilation (via increased nitratereductase activity) (Bahuguna et al., 2012).

Pathophysiology and EpidemiologyThe central role of B1 in central (oxidative) metabolismin humans is reflected in its pathophysiology upon vitamindeficiency. TPP plays an indispensable role in energy metabolismas a cofactor in cleavage of α-keto acids (Adeva-Andany et al.,2017), as well as general oxidative metabolism, identical toits role in planta. While having lost the ability to synthesizethiamin during their evolution, humans possess the potential tointerconvert the different thiamin phosphate-esters (Zhao et al.,2001; Banka et al., 2014).

B1 was the first vitamin for which deficiency wascharacterized, as it was considered a “vital amine’ (hence‘vitamine’), defined as a substance inducing the disease beriberiupon insufficient consumption (Lonsdale, 2006). Beriberi isa disease occurring upon severe B1 deficiency, divided in wetand dry beriberi, depending on whether it is manifested inthe cardiovascular system or in the peripheral nervous system,respectively (Abdou and Hazell, 2015). B1 deficiency can causeheart problems, and even lead to heart failure (Roman-Camposand Cruz, 2014). Different symptoms, such as enlarged heart andincreased venous pressure, have been reported. B1 deficiencyhas also been linked to Sudden Infant Death Syndrome (SIDS),due to brainstem malfunctioning related to hypo-oxidativemetabolism (Lonsdale, 2015). An insufficient supply of theB1-vitamin can induce severe alternation of the nervous system,which leads to a disorder called Wernicke’s encephalopathy

(WE) (Jung et al., 2012). WE involves the arising of selectivebrain lesions, the first symptoms of which include confusion,apathy and impaired awareness, eventually ending in comaand death. B1 deficiency-induced disorders are in many caseseasily reverted with thiamin application, and often witnessed inpatients suffering from chronic alcoholism (Butterworth, 1993).The detrimental effect of B1-deficiency on brain functioningcan be explained by the strong dependency of the brain on theoxidative metabolism (Butterworth, 1993; Gibson et al., 2005).

One of the greatest risks of B1-deficiency, along with the lethalconsequences of untreated WE, is the difficulty of diagnosis,leaving many illnesses untreated (Harper, 2006). Althoughcases of severe beriberi have become rare, outbreaks of B1-deficiency-induced beriberi have been reported on a global scale,causing many deaths, even upon sufficient access to healthcare(Luxemburger et al., 2003; Ahoua et al., 2007). Hence, indeveloping countries, B1-deficiency often is not linked to theobserved casualties (Barennes et al., 2015). Moreover, infantileexposure to B1-deficiency was recently shown to have long-term effects on motor functions and balance of the child (Harelet al., 2017). Furthermore, elderly people have been shown to behighly susceptible to B1-deficiency, even in the developed world(Hoffman, 2016). Indeed, an investigation in New York state(United States) identified 14% of elderly as being B1-deficient(Lee et al., 2000). B1-deficiency is likely to be exacerbatingAlzheimer’s disease, and could therefore be considered a seriousthreat, definitely not confined to the developing world (Gibsonet al., 2013).

Good sources of vitamin B1 are, besides animal-derivedproducts (meats, liver, eggs, and dairy products), beans andpeas, nuts and whole grains (Lonsdale, 2006; USDA, 2016).Different massively consumed crops, such as rice, cassava, potatoand plantain contain inadequate amounts of B1 (Table 1). Inthe case of rice, polishing, which removes the aleurone layerto avoid rancidification, eliminates many nutritionally valuablesubstances, including B1 (Goyer, 2017). This is illustrated by theoriginal observation of B1-deficiency induced paralysis and deathin fowls fed with polished rice, reversible by administration of therice polishings (Lonsdale, 2006). Therefore, overconsumption ofsuch staples in a monotonous diet, imposes a serious threat tohuman health. Furthermore, high carbohydrate intake increasesthe need of dietary B1, which is explained by its role incarbohydrate catabolism (Elmadfa et al., 2001). This emphasizesthe need for increased B1 levels in these popular starchy crops.

BiofortificationEngineering of the thiamin biosynthesis pathway to augmentthiamin content in plants has been attempted recently (Donget al., 2015, 2016). The key step in thiamin -and therefore B1-engineering is the first committed step in plastidial pyrimidinebiosynthesis, THIC (Raschke et al., 2007). Activity of the THICenzyme seems to be a major determinant of B1 biosynthesis,as indicated by the oscillations of the corresponding mRNAtranscript with TMP levels (Bocobza et al., 2013). Moreover,this gene harbors a TPP-binding riboswitch in its 3′ UTR,which enables it to destabilize its mRNA upon high TPPprevalence (Wachter et al., 2007). This feedback mechanism,

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rather unique in eukaryotes, further highlights THIC as aregulatory point in B1 biosynthesis and therefore the THICgene as an ideal candidate in metabolic engineering approaches(Pourcel et al., 2013). Indeed, eliminating this riboswitch, therebyremoving the feedback inhibition on THIC, elevates thiaminlevel 1.6-fold in Arabidopsis (Bocobza et al., 2013). Enhancingthe flux toward biosynthesis of the pyrimidine intermediateis likely insufficient for accumulation of B1. Indeed, feedingArabidopsis seedlings with the intermediates pyrimidine andthiazole indicates that both are necessary to achieve higherlevels of B1 (Pourcel et al., 2013). Combined overexpressionof THIC and THI1, the plastidial thiazole biosynthetic enzyme(Godoi et al., 2006), further enhanced B1 levels of Arabidopsisover threefold compared to wild type (Dong et al., 2015).Similarly, implementation of this combined engineering strategyin rice resulted in B1 increase of 2.5-fold in leaves and5-fold in unpolished grains (Dong et al., 2016). However, B1levels remained barely affected in polished rice seeds. Futureengineering strategies in B1-biofortification will tackle additionalbottlenecks in B1-accumulation as well as applying engineeringstrategies to specific tissues (Goyer, 2017). Taken into account thedetrimental effects of THIC-riboswitch elimination, resulting inchlorotic plants with enhanced carbohydrate oxidation (Bocobzaet al., 2013), B1 biofortification should be approached withcaution.

Besides metabolic engineering, there are opportunities toenhance B1 content in crops via breeding techniques. Indeed,several (wild) potato varieties were identified which harbor over2-fold difference in B1 content compared to popular agriculturalpotato cultivars (Goyer and Sweek, 2011). Similarly, up to 2.7-foldvariation was found in different cassava accessions (Mangel et al.,2017). Previously, over 10-fold B1 variation has been measured inrice (Kennedy and Burlingame, 2003). Recently, a genome wideassociation study (GWAS) identified multiple quantitative traitloci (QTL), underlying B1 content in common wheat (Li et al.,2017). These results imply that breeding strategies could help inacquiring higher B1 levels in popular/regional crop varieties. Onthe other hand, elevating of B1 levels through exposure to certainbiological stresses has been suggested, as this proves to augmentB1 biosynthesis by significantly increasing the expression of thebiosynthesis genes (Kamarudin et al., 2017).

VITAMIN B6

Vitamin B6 represents a group of water-soluble molecules withsimilar biochemical properties, consisting of pyridoxine (PN),pyridoxal (PL), pyridoxamine (PM), and their phosphorylatedesters (Fudge et al., 2017). PN, PL and PM differ bycarrying a hydroxymethyl, an aldehyde or an aminomethylsubstituent, respectively (Figure 2) (Hellmann and Mooney,2010). Considering these six vitamers, the phosphorylatedpyridoxal (PLP, Figure 2D) is the most bioactive, functioning as acofactor in over a hundred reactions (Fudge et al., 2017). B6 canbe considered a powerful antioxidant, comparable to carotenes(vitamin A) and tocopherols (vitamin E), as they are able toquench ROS (Bilski et al., 2000).

FIGURE 2 | Chemical structure of different B6 vitamers. (A) pyridoxal (PL), (B)pyridoxine (PN), (C) pyridoxamine (PM), (D) pyridoxal-phosphate (PLP), (E)pyridoxine-phosphate (PNP), (F) pyridoxamine-phosphate (PMP).

BiosynthesisDe novo biosynthesis of vitamin B6 takes place in the cytosoland comprises only two enzymes (Figure 3). Pyridoxal phosphatesynthase protein (PDX1) generates pyridoxal 5′-phosphate(PLP) utilizing ammonia, glyceraldehyde 3-phosphate (G3P)and ribose 5′-phosphate (R5P) as substrates (Titiz et al., 2006).This ammonia originates from the reaction catalyzed by thePDX2 glutaminase, which releases ammonia from glutamine toyield glutamate (Tambasco-Studart et al., 2007). Furthermore,PMP/PNP oxidase (PDX3) is considered a crucial step in PLPsalvage, ensuring its retrieval from the PMP and PNP vitamers(Sang et al., 2007). The non-phosphorylated vitamers PL, PM,and PN, can be converted to their corresponding phosphorylatedvitamers by the action of the SALT OVERLY SENSITIVE 4 kinase(SOS4) (Shi et al., 2002). Finally, a pyridoxal reductase (PLR1)was identified, mediating a NADPH-requiring conversion of PLto PN (Herrero et al., 2011). Through these reactions plants arecapable of balancing the different vitamer forms of B6, which isrequired to ensure controlled growth and development (Colinaset al., 2016).

Role in Plant PhysiologyVitamin B6 is involved in a plethora of metabolic reactions,serving as cofactor or required as an antioxidant (Tambasco-Studart et al., 2005; Mooney and Hellmann, 2010). PLP isconsidered to function as a cofactor for about 200 enzymaticreactions in Arabidopsis (Fudge et al., 2017). These PLP-dependent enzymes, covering oxidoreductases, transferases,hydrolases, lyases, and isomerases, can be explored using the B6database tool (Percudani and Peracchi, 2009). These reactionsroughly cover the whole spectrum of plant metabolism. In doingso, B6 is required in amino acid synthesis as well as catabolism(Mooney and Hellmann, 2010). This is illustrated by theArabidopsis mutant reduced sugar response (rsr4-1), harboring

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FIGURE 3 | Vitamin B6 biosynthesis in plants. De novo biosynthesis of vitamin B6 is cytosolic. PLP, the major bioactive B6 vitamer is synthesized via consecutiveaction of PDX2 and PDX1 enzymes. Reactions required to interconvert these different vitamers are depicted. Biosynthesis pathway is shown in blue, enzymes inblack. Products: Gln, glutamine; Glu, glutamate; NH3, ammonia; R5P, ribose 5′-phosphate; G3P, glyceraldehyde 3-phosphate; PL, pyridoxal; PN, pyridoxine; PM,pyridoxamine; PLP, pyridoxal phosphate; PNP, pyridoxine phosphate; PMP, pyridoxamine phosphate; NADPH, nicotinamide adenine dinucleotide phosphate.Enzymes: PDX1, pyridoxal phosphate synthase protein; PDX2, pyridoxine biosynthesis glutaminase; PDX3, PMP/PNP oxidase; SOS4, SALT OVERLY SENSITIVE4;NSP, non-specific phosphatase; PLR1, pyridoxal reductase.

a mutated B6 biosynthesis gene (PDX1), exhibiting a decreasedcontent of shikimate, altered levels of different amino acids, andhigher levels of TCA constituents (malate, citrate and fumarate)(Wagner et al., 2006). Similarly, Arabidopsis mutants for the PLPsalvage enzyme PDX3 (involved in B6 vitamer interconversions)contained aberrant amino acid profiles. The initial step in starchbreakdown, α-glucan phosphorylase, requires PLP as a cofactor(Mooney and Hellmann, 2010). Furthermore, PLP-dependentenzymes play a role in synthesis of glucosinolates (Mikkelsenet al., 2004). Remarkably, biosynthesis of the plant hormonesauxin (Zhao, 2010) and ethylene (Van de Poel and Van DerStraeten, 2014; Vanderstraeten and Van Der Straeten, 2017) aswell as ethylene breakdown (Nascimento et al., 2014) involvePLP-requiring enzymes. B6 levels have also been linked tonitrogen metabolism as pdx3 lines were shown to be ammoniumdependent (Colinas et al., 2016). This link is further strengthenedby the observation that the ammonium transporter mutant amt1has altered B6 levels (Pastor et al., 2014).

On top of its vast influence on plant metabolism via PLP-depending enzymes, B6 plays a crucial role as an antioxidant(Vanderschuren et al., 2013; Fudge et al., 2017). Arabidopsismutants with a lowered B6 status, exhibit distinct phenotypesincluding poor seed development, delayed flowering and

reduced plant growth, while complete knock-outs are lethal(Vanderschuren et al., 2013). The lowered tolerance of thesemutants to salt, high light, ultraviolet light, and oxidativestress illustrate the importance of B6 as a stress protector(Vanderschuren et al., 2013). Upon heat stress, a non-catalyticpyridoxine biosynthesis protein (PDX1.2), ensures sufficientB6 production by aiding its paralogs (PDX1.1 and PDX1.3),resulting in an increase of B6 content (Moccand et al., 2014;Dell’Aglio et al., 2017). Conversely, Arabidopsis lines, engineeredfor enhanced B6 content, display enhanced tolerance to abioticstresses (Raschke et al., 2011). Furthermore, these plants exhibitenlarged cells, leading to larger organs. Interestingly, their aminoacid and sugar composition is severely altered, reflecting thebroad influence of B6 on plant metabolism.

Pathophysiology and EpidemiologyVitamin B6, especially PLP, is crucial for correct humanfunctioning, as it is required as a cofactor for around 4%of all enzyme activities (Ueland et al., 2017). Most of thesereactions involve amino acid synthesis and catabolism, in whichPLP serves as a cofactor in transaminations, aldol cleavagesand carboxylations. Furthermore, PLP plays a role in energymetabolism as it is involved in gluconeogenesis and lipid

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metabolism. Moreover, B6 is necessary in the biosynthesis ofheme as well as neurotransmitters (Ueland et al., 2017). Inaddition, B6 plays an important role as an antioxidant (Justinianoet al., 2017) and is even known to aid in enzyme folding (Celliniet al., 2014).

In parallel with its functions in human metabolism, B6deficiency is manifested in a broad spectrum of disorders.Most notably, B6 deficiency is known to provoke neurologicaldisorders, such as peripheral neuropathy (Ghavanini andKimpinski, 2014) and epileptic seizures (Skodda and Muller,2013). Moreover, B6 deficiency might be linked to anemia, giventhe ability of B6 intake to cure some cases of the disease (Hisanoet al., 2010). Furthermore, B6 deficiency has been associated withcardiovascular diseases, stroke, rheumatoid arthritis, diabetes anddifferent types of cancer including colorectal, lung, breast, andkidney (Ueland et al., 2017).

Although investigation on vitamin B6 deficiency on a globalscale is lacking, there is evidence supporting the existence ofpersistent deficiency in several populations (Fudge et al., 2017).Indeed, studies in the United States and South Korea concludedthat around one-in-four people have sub-optimal B6 status(Pfeiffer et al., 2013; Kim and Cho, 2014). Furthermore, halfof the elderly in nursing homes in Norway were consideredB6 deficient (Kjeldby et al., 2013). The situation in developingcountries is estimated to be even worse, given the observationthat over half of the population of Uganda and Sudan remain B6deficient (Fudge et al., 2017). Knowing the detrimental effect thisdeficiency, remaining undiagnosed, could exert on human health,there is a strong need to supply these people with satisfactoryamounts of B6.

Humans, unable to synthesize B6 de novo, predominantlydepend on their diet for sufficient B6 acquisition, as gut bacteriacan be considered as suppliers of marginal amounts of differentvitamins (LeBlanc et al., 2013; Fudge et al., 2017). Good sourcesof dietary B6, besides animal-derived products such as fish andmeat, are fresh vegetables including carrots and onions (USDA,2016; Fudge et al., 2017). However, bioavailability should beconsidered, given the observations that up to half of the B6 poolcould be lost as a result of incomplete digestibility, which is foundto be more problematic in plant-based food sources compared toanimal products (Roth-Maier et al., 2002). Furthermore, the mostconsumed staple crops in the world are considered poor sourcesof dietary B6 (Fudge et al., 2017) (Table 1).

BiofortificationMetabolic engineering approaches rely on the knowledgeacquired of the relatively simple plant B6 biosynthesis pathway,mainly involving PDX2 (Tambasco-Studart et al., 2007) and thepyridoxal phosphate synthase protein (PDX1) (Titiz et al., 2006).In a metabolic engineering strategy, overexpression of both PDX1and PDX2 genes yielded up to fourfold increase in B6 levels, whileoverexpression of the single genes only generated marginal effects(Raschke et al., 2011). Interestingly, enhanced plant biomass inaerial organs with similar overall morphology as well as toleranceto oxidative stress were observed in two-gene engineered plantswith increased B6 content. When targeted to roots, the two-geneapproach, enabled almost sixfold augmentation of B6 in cassava,

without any severe alteration in yield (Li et al., 2015). Thesuccess of this two-gene engineering strategy therefore supportsassessment in different crops, as well as investigation of possibleinfluences on crop physiology and yield.

So far, analysis of crop germplasm has revealed limitedvariation (<2-fold) in B6 composition of potato (Mooney et al.,2013) and wheat (Shewry et al., 2011). However, screening of vastaccessions of a particular crop could identify interesting lines andthereby also pinpoint novel important QTLs and maybe novelgenes influencing B6 homeostasis (Fudge et al., 2017).

VITAMIN B9

Folate is a collective term for a group of water soluble B9 vitamins.Folates can be considered tri-partite structures, consisting of apterin ring linked to the para-aminobenzoate (p-ABA) moietycarrying a γ-linked glutamate tail (Scott et al., 2000; Rebeilleet al., 2006) (Figure 4). The different folate species, calledvitamers, are chemically different on three levels, being theoxidation state, the glutamate tail length and the nature ofC1-substituents (Blancquaert et al., 2010; Strobbe and Van DerStraeten, 2017). These properties all exert an influence on folatestability. First, oxidized folates are considered more stable, giventhe susceptibility of the pterin – p-ABA linkage to (photo-)oxidative cleavage (Blancquaert et al., 2010). Tetrahydrofolates(THF), the most reduced folate forms, harboring a fully reducedB-ring in the pterin moiety, are the active cofactors. Conversely,folic acid, containing an aromatic pterin B-ring, is more stable,though exhibiting marginal natural occurrence (Blancquaertet al., 2010; Gorelova et al., 2017b). In this respect, the term ‘folicacid’ is used to indicate the synthetic folate analog. Second, folateentities greatly differ in their glutamate tail length, as they carryone to eight glutamates (Garratt et al., 2005; Strobbe and Van DerStraeten, 2017). Polyglutamylated folates are thought to possessenhanced in vivo stability as their polyglutamate tail ensurescellular retention as well as augmented association with folatedependent enzymes (Blancquaert et al., 2014). Third, folatesspecies can differ in their attached C1- units, giving rise to anarray of folate entities, affecting their stability and biological role(Figure 4).

BiosynthesisIn plants, folate biosynthesis is executed in different subcellularlocalizations (Figure 5). The pterin ‘branch’ resides in the cytosol(Strobbe and Van Der Straeten, 2017). Here, the first committedstep is executed by GTP cyclohydrolase I (GTPCHI), utilizingGTP as a substrate and yielding 6-hydroxymethyldihydropterin(HMDHP) (Basset et al., 2002). An alleged pterin mitochondrialimporter is considered to ensure translocation of HMDHP tothe mitochondrion (Hanson and Gregory, 2011; Strobbe andVan Der Straeten, 2017). The plastidial p-ABA branch suppliesthe p-ABA moiety of the folate molecule (Figure 5). Here, thefirst committed step is performed by aminodeoxychorismatesynthase (ADCS), using chorismate, originating from theshikimate pathway (Herrmann and Weaver, 1999), as a substrate(Sahr et al., 2006). Given the hydrophobic nature of p-ABA,

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FIGURE 4 | Chemical structure of folates (B9). The three different folate components, pterin (green), para-aminobenzoate (blue), and glutamate tail (red) areindicated. Here, a fully reduced tetrahydrofolate (THF) is presented. Figure adopted from (Strobbe and Van Der Straeten, 2017).

it is thought to reach the mitochondria by diffusion throughmembranes (Hanson and Gregory, 2011; Strobbe and Van DerStraeten, 2017). Upon entering the mitochondria, HMDHPis pyrophosphorylated and coupled with p-ABA to formdihydropteroate. These enzymatic reactions are executed bythe bifunctional HMDHP pyrophosphokinase/dihydropteroatesynthase (HPPK/DHPS) (Gorelova et al., 2017a). Subsequently,dihydropteroate is converted to dihydrofolate (DHF) by theaction of dihydrofolate synthetase (DHFS) (Ravanel et al.,2001), followed by a reduction catalyzed by dihydrofolatereductase as part of a bifunctional enzyme dihydrofolatereductase/thymidylate synthase (DHFR-TS) (Gorelova et al.,2017b), yielding THF. Folate biosynthesis is finalized uponpolyglutamylation of THF, by the action of folylpolyglutamatesynthetase (FPGS) (Ravanel et al., 2001; Mehrshahi et al., 2010).

Role in Plant PhysiologyThe chemical structure of folates makes them ideal carriers ofC1-substituents, conferring a central role in carbon metabolismof nearly all living organisms (Blancquaert et al., 2010), withthe exception of some Archaea (Gorelova et al., 2017b).Thereby, folates are both needed for proper anabolism as wellas catabolism of cellular compounds. They play an essentialrole in the synthesis of purines as well as thymidylate and aretherefore indispensable in DNA synthesis and growth (Stover,2004). Furthermore, folates are required in biosynthesis ofmany plant metabolites including pantothenate (vitamin B5)and formyl methionyl tRNA as well as serine and glycineinterconversion and catabolism of histidine (Blancquaert et al.,2010). Furthermore, folates are needed in production of lignin,ensuring cell wall rigidity (Srivastava et al., 2015). In addition,iron-sulfur cluster enzymes depend on folates for their assembly

(Waller et al., 2010). Given their role as C1 donors and acceptors,folates play a key role in the methyl cycle (Blancquaert et al.,2010). 5-methyl-THF, is required as a methyl-donor in theconversion of homocysteine to methionine, which is necessaryfor replenishing of the SAM-pool (Blancquaert et al., 2010).SAM in its turn, functions as methyl storage in supplying thisC1-unit to a wide range of methyltransferases, including DNAmethyltransferases. Therefore, insufficient folate can alter themethyl-cycle homeostasis and evoke epigenetic changes byalteration in the DNA methylation pattern (Zhou et al., 2013).A disequilibrated folate homeostasis greatly influences epigeneticfunctioning through genome-wide hypomethylation, loweredhistone methylation and transposon derepression, as witnessedin Arabidopsis methyleneTHF dehydrogenase/methenylTHFcyclohydrolase (MTHFD1) mutants (Groth et al., 2016).Similarly, aberrant functioning of FPGS, the enzyme responsiblefor extension of the glutamate tail, evoked upregulation oftransposable elements (typically repressed by methylation),which could be reverted via administration of 5-methyl-THF(Zhou et al., 2013).

Additional to their requirement in catabolism and anabolismof essential plant metabolites, folates appear to have aprofound influence on plant growth and development. Innon-photosynthetic plastids, the plastidial pool of folatesinfluences plant energy metabolism by inhibiting starchformation (Hayashi et al., 2017). The mechanism is thoughtto operate via depletion of the ATP pool -required in starchassembly from sucrose- upon folate shortage, regulated by thefolate-dependent DHFR-TS (Hayashi et al., 2017). Remarkably,the interplay of folate and sugar metabolism was shown tomodulate auxin signaling, hence controlling plant development(Stokes et al., 2013). Moreover, folates possess the ability

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FIGURE 5 | Folate biosynthesis is plants. Folate (vitamin B9) biosynthesis in plants occurs in three subcellular compartments: the cytosol, the plastids (green) andthe mitochondrion (red). Biosynthesis pathway is shown in blue, enzymes in black. Polyglutamylated folates are considered the end product of folate biosynthesis.Products: ADC, aminodeoxychorismate; p-ABA, para-aminobenzoate; DHN-P3, dihydroneopterin triphosphate; DHN-P, dihydroneopterin monophosphate; DHN,dihydroneopterin; HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, HMDHP pyrophosphate; DHP, dihydropteroate; DHF, dihydrofolate; Glu, glutamate; THF,tetrahydrofolate. Enzymes: ADCS, ADC synthase; ADCL, ADC lyase; GTPCHI, GTP cyclohydrolase I; DHNTPPH, dihydroneopterin triphosphatepyrophophohydrolase; NSP, non-specific phosphatase; DHNA, DHN aldolase; HPPK, HMDHP pyrophosphokinase; DHPS, DHP synthase; DHFS, DHF synthetase;DHFR, DHF reductase; FPGS, folylpolyglutamate synthetase.

to influence seed composition, demonstrated by the highN-content of Arabidopsis plastidial FPGS (atdfb-3) loss-of-function mutant seeds (Meng et al., 2014). This reveals aninteraction between folate metabolism and N-metabolismin darkness. Folate metabolism was also shown to maintainroot development in the indeterminate state, via FPGSfunctioning (Reyes-Hernandez et al., 2014). Folate synthesisand therefore accumulation is high during germination andin meristematic tissues, coherent with their demand uponcell division and concomitant DNA synthesis (Rebeille et al.,2006). Moreover, folate biosynthesis is stimulated upon lightexposure, indicating a higher folate requirement (Rebeille et al.,2006). Indeed, the production of chlorophyll is dependenton folate (Van Wilder et al., 2009). Moreover, folates are able

to ensure sufficient NADPH production, thereby controllingcellular redox state by a balanced functioning of DHFR-TSgenes, needed in detoxification of ROS originating fromphotosynthesis or photorespiration (Gorelova et al., 2017b). Inphotorespiration, folate is directly required as a cofactor for theserine hydroxymethyltransferase in the glycine decarboxylasecomplex (Collakova et al., 2008; Maurino and Peterhansel, 2010).Finally, folate biosynthesis enzymes are known to influenceplant stress responses, possibly through generation of folatebiosynthesis intermediates (Storozhenko et al., 2007b; Navarreteet al., 2012).

Given the influence of folates on plant development, theirhomeostasis and accumulation is considered to be tightlyregulated, depending on their tissue specific requirement

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(Rebeille et al., 2006). Indeed, recent insights in folatemetabolism of Arabidopsis confirm fine-tuning of folateaccumulation by feed-back inhibition of a regulatory DHFR-TShomolog (DHFR-TS3) (Gorelova et al., 2017b). Together, thesefindings raise caution toward possible implications upon folatebiofortification, as an increased folate pool might influencedifferent aspects of plant physiology (Van Wilder et al., 2009).

Pathophysiology and EpidemiologyHumans lack the ability to synthetize folates de novo. However,they possess DHFR and FPGS enzymes, thereby allowingconversion of DHF to THF and polyglutamylated folates,respectively (Masters and Attardi, 1983; Garrow et al., 1992).Hence, humans are almost completely reliant on their diet foradequate folate supply, given that the gut microbiome has amarginal contribution to the folate pool (Camilo et al., 1996;LeBlanc et al., 2013). As the usage of folates as C1 donorsand acceptors originated early in evolution, being implementedby prokaryotes and all eukaryotes, their basic functioning inplants is very similar to that in humans. Thus, folates areimportant in DNA synthesis and in supplying methyl groupsto proteins, lipids, and DNA, through their necessity in SAMreplenishment (Saini et al., 2016). Similar to plants, changes infolates levels have the potency to change the human epigenome(Bistulfi et al., 2010). Folates are required in methylation ofmyelin basic protein, which is pivotal for the compaction ofmyelin around the neuron sheath, thereby ensuring sufficientnerve conduction (Ramaekers and Blau, 2004; Bottiglieri,2005).

Upon inadequate dietary folate intake, folate status candrop, a condition known as folate deficiency, which has abroad pathophysiology. Folate deficiency results in decreasederythrocyte development, causing megaloblastic anemia(Lanzkowsky, 2016). The elevated levels of homocysteine,resulting from low folate status, can induce vascular diseases,such as coronary artery disease and strokes (Antoniadeset al., 2009; Guo et al., 2009; Zeng et al., 2015). The mostnotable consequence of folate deficiency is its detrimentalimpact on neurulation. This is revealed by the occurrence ofneural tube defects (NTDs) such as spina bifida, encephaloceleand anencephaly, caused by folate deficiency (Geisel, 2003;Youngblood et al., 2013; Greene and Copp, 2014). Last but notleast, different forms of cancer have been linked to inadequatefolate status, including colorectal (Feng et al., 2017), prostate(Price et al., 2016), and pancreatic tumors (Yallew et al., 2017).

Folate deficiency is still a global problem, predominantlypresent in the developing world, yet persisting in manypopulations of the developed world as well (Blancquaert et al.,2014; Zaganjor et al., 2015). Moreover, even populations blessedby the availability and opportunity of a diverse and folate-rich diet, remain susceptible to deficiency, as illustrated by thelow folate status measured in the Swedish population (Eussenet al., 2013; Gylling et al., 2014) and the observed sub-optimalfolate levels in 39% of Belgian first trimester pregnancies(Vandevijvere et al., 2012). Worldwide, 300,000 pregnancies areestimated to be affected by NTDs annually, half of which areconsidered to be caused by insufficient maternal folate status

(Flores et al., 2014). China, inhabited by almost 1.4 billionpeople, recorded a countrywide prevalence of NTDs as highas 0.24% (Blancquaert et al., 2014). More strikingly, Shanxiprovince, located in Northern China, has amongst the highestincidence rates of NTDs in the world, as high as 1.39% (Li et al.,2006).

Fortunately, noteworthy advances have been made in thefight against folate malnutrition. Educational efforts, advocatinga diverse diet containing folate rich foods such as green leafyvegetables and fermented products, is the primary strategy todiminish folate deficiency (Strobbe and Van Der Straeten, 2017).Folic acid, the synthetic form of folate as administered inpills, has been implemented in fortification strategies, whichhave ensured a significant reduction of neural tube defects(Williams et al., 2015; Wang et al., 2016). Unfortunately,high folic acid intake can also impose unwanted side effects,since excessive accumulation of unmetabolized folic acid hasbeen linked to colorectal cancer and impaired immunity (Choet al., 2015; Selhub and Rosenberg, 2016). Moreover, both folicacid fortification and supplementation are costly interventions,which are difficult to implement in poor rural regions inneed (Blancquaert et al., 2014). Therefore, biofortification, viametabolic engineering or breeding is advised to ensure a stablecost-effective means to fight folate deficiency (De Steur et al.,2012, 2015; Blancquaert et al., 2014; Strobbe and Van DerStraeten, 2017).

BiofortificationOver the last decades, many successful folate biofortificationapproaches have been conducted, thereby additionally acquiringnew insights in folate metabolism in certain crops and tissues(De Lepeleire et al., 2017; Strobbe and Van Der Straeten,2017). The most widely attempted folate metabolic engineeringapproach is the enhancement of GTPCHI activity, proven to bea fruitful strategy in prokaryotes (Sybesma et al., 2003). Thisapproach has been confirmed to be functional in plants bythe engineering of cis-genic Arabidopsis lines, over-expressingGTPCHI (Hossain et al., 2004). This single gene approach,introducing GTPCHI, referred to as G-engineering, has beenimplemented in rice (Storozhenko et al., 2007a), tomato (de laGarza et al., 2004), maize (Naqvi et al., 2009), lettuce (Nuneset al., 2009), potato (Blancquaert et al., 2013a), and Mexicancommon bean (Ramírez Rivera et al., 2016). The highest foldenhancement, reached in the edible portions of these crops is aninefold folate increase in lettuce. This could possibly be due toa difference in regulation in leafy tissue. However, single geneapproaches have hitherto not resulted in over 10-fold increasein folate content. A bigenic approach was substantially moresuccessful, adding ectopic expression of aminodeoxychorismatesynthase (ADCS) (GA-strategy). In tomato (de la Garza et al.,2007) and rice (Storozhenko et al., 2007a) this led to 25- and100-fold folate enhancement, respectively. Unfortunately, thisapproach, able to reach the desired levels in tomato and rice,does not promise to be universally applicable, as it only resultedin limited enhancements in Arabidopsis and potato (Blancquaertet al., 2013a). In rice seeds, ADCS has been indicated as the mostimportant limiting factor in folate biosynthesis, additional to

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GTPCHI (Dong et al., 2014a). Building further on these findings,novel biofortification approaches aimed at further gene stacking,using mitochondrial folate biosynthesis genes (Strobbe and VanDer Straeten, 2017). Indeed, additional introduction of FPGSin GA-engineered plants did not only result in elevated folatelevels in rice endosperm (100-fold) and potato tubers (12-fold)respectively, but also in enhanced folate stability upon storage(Blancquaert et al., 2015; De Lepeleire et al., 2017). Increasingstorage stability has also been addressed by introduction ofmammalian folate binding proteins (Blancquaert et al., 2015).This strategy is promising, as it could limit the aforementionedundesired effects of folate increase on plant physiology, viasequestration of the active folate pool. Moreover, recent discoveryof plant folate binding proteins creates novel opportunities infolate biofortification via metabolic engineering (Puthusseri et al.,2018).

Breeding endeavors, aimed at acquiring elite crop variantswith augmented folate content in the edible portion, thoughnot implemented so far, have shown to be feasible (Anderssonet al., 2017; Bouis and Saltzman, 2017). Upon availability of highthroughput folate quantification in the food matrix, screeningof vast germplasm collections could lead to identification ofhigh folate varieties (De Brouwer et al., 2010; Strobbe and VanDer Straeten, 2017). In this respect, over sevenfold variation inmilled rice folate content was described by examination of 78accessions (Dong et al., 2011). More recently, unpolished brownrice folate content was found to vary up to threefold in 150examined accessions (Aiyswaraya et al., 2017). Similar screening

has been employed in barley (Andersson et al., 2008), red beet(Wang and Goldman, 1996), potato (Goyer and Sweek, 2011;Robinson et al., 2015), tomato (Iniesta et al., 2009), muskmelon(Lester and Crosby, 2002), common bean (Khanal et al., 2011;Jha et al., 2015), lentil (Jha et al., 2015), (chick)pea (Jha et al.,2015), spinach (Shohag et al., 2011), and strawberry (Mezzettiet al., 2016). Furthermore, these variations could be utilized toidentify interesting QTLs, underlying folate content, in GWAS(Khanal et al., 2011; Dong et al., 2014b). These techniques, thoughlimited in their potential folate enhancement, are promising, asthey might face lower regulatory restrictions, hence allow morerapid implementation in agriculture, reaching the populations inneed (Mejia et al., 2017; Potrykus, 2017).

B-VITAMIN INTERPLAY

Multi-biofortification is considered an important goal in thefight against MNM (Blancquaert et al., 2014; Strobbe andVan Der Straeten, 2017). However, possible effects of alteredmicronutrient levels upon each other as well as on basic plantgrowth and development, should be taken into consideration.Examination of the role of B-vitamins in plant metabolismevidently reveals that inducing their accumulation could alterplant physiology. This has been conspicuously observed inmetabolic engineering approaches of B1 (Bocobza et al., 2013;Dong et al., 2015) and B6 (Raschke et al., 2011). Furthermore,B9 enhancement, though not depicting any severe effect on

FIGURE 6 | B-vitamin interplay in planta. Different interactions of vitamin B1 [via thiamin pyrophosphate (TPP) functioning], B6 [via pyridoxal phosphate (PLP)functioning] and B9 (folates) are schematically displayed. Well-established links are indicated in black, potential interactions are indicated in blue. TCA, tricarboxylicacid cycle; PPP, pentose phosphate pathway; G3P, glyceraldehyde 3-phosphate; R5P, ribose 5′-phosphate; SAM, S-adenosylmethionine; AIR, 5-aminoimidazoleribonucleotide; NADPH, nicotinamide adenine dinucleotide phosphate.

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plant growth, has shown to alter the rice seed metabolism(Blancquaert et al., 2013b). The influence of B-vitamins on plantmetabolism is, however, at least partly intertwined, indicating theimportance of detailed investigation of the effect of theircombined biofortification (Figure 6).

In central energy metabolism, both folate and B1 appearto negatively influence the plant’s ability to accumulate starch(Bocobza et al., 2013; Hayashi et al., 2017). PLP (B6) is alsoinvolved in starch breakdown, though there are no indicationsto suspect increased starch breakdown upon elevation of PLPlevels (Zeeman et al., 2004; Mooney and Hellmann, 2010).In the biosynthesis of B6, G3P, an intermediate in centralenergy metabolism (glycolysis), serves as a substrate (Fudgeet al., 2017), the steady state concentration of which mightbe altered in B1 engineered lines (Bocobza et al., 2013). Inaltering this central metabolism equilibrium, B1 augmentationmight influence the flux through the shikimate pathway (Bocobzaet al., 2013), the activity of which is required in the plastidialpart of folate biosynthesis (Strobbe and Van Der Straeten,2017). In this shikimate pathway, PLP (B6) is required ascofactor. Folates are able to generate NADPH (Gorelova et al.,2017b), replenish the SAM pool (Blancquaert et al., 2010), andare needed in the biosynthesis of iron sulfur cluster enzymes(Waller et al., 2010). Interestingly, THIC, pinpointed as therate limiting step in B1 biosynthesis, contains an iron-sulfurcluster and requires SAM for its catalytic activity (Pourcel et al.,2013). Strongly increased THIC activity would therefore requireenhanced SAM turnover (Palmer and Downs, 2013), for whichenhanced folate levels might have a beneficial effect. NADPHis on the other hand required for pyridoxal reductase activityin B6 homeostasis. Ribose 5′-phosphate, an important substratein B6 biosynthesis (Fudge et al., 2017), is a product of thepentose phosphate pathway, the flux of which might be controlledby B1 (Bocobza et al., 2013). Similarly, AIR, an importantsubstrate in B1 biosynthesis (Pourcel et al., 2013), is derivedfrom purine metabolism, the synthesis of which is dependenton folate (Strobbe and Van Der Straeten, 2017). Moreover, B1,B6, and B9 have been linked to nitrogen metabolism. First,thiamin application is known to stimulate nitrogen assimilation(Bahuguna et al., 2012). Second, B6 content was observed to bealtered in the ammonium transporter mutant amt1 (Pastor et al.,2014). Moreover, PLP (B6) salvage mutant pdx3 is dependingon ammonium (Colinas et al., 2016). Third, folate biosynthesismutants (atdfb-3, plastidial FPGS) harbored enhanced nitrogencontent of seeds (Meng et al., 2014). Remarkably, given thelabile nature of folate, increasing in planta stabilization of folateshas been the subject of biofortification strategies (Blancquaertet al., 2015). Therefore, enhancing levels of antioxidants, such asB6, has been proposed as an additional biofortification strategy,protecting the folate pool from oxidative cleavage (Blancquaertet al., 2014).

FINAL REMARKS

Creation and evaluation of multi-biofortified crops would notonly offer a sustainable solution to eradicate MNM, but alsohelp to elucidate the interplay of different micronutrients. Theavailability of novel tools, allowing facilitated cloning of multiplegenes paved the way toward such multi-biofortification (Engleret al., 2014). Furthermore, a prerequisite in biofortificationstrategies is to consider stability upon storage of the crop product,as well as after food processing and bioavailability upon humanconsumption (Blancquaert et al., 2015; Diaz-Gomez et al., 2017).Different agronomical techniques could be employed, alone orin combination, to augment vitamin content of crops. Metabolicengineering of the complete pathway, or symbiosis with bacteria,might be appropriate ways to tackle vitamin B12 deficiency(DeMell and Holland, 2016). Metabolic engineering strategiescould be developed in a precise way, enabling the creation offood crops which harbor an ideal balance of energy supply andmicronutrient delivery, while exhibiting marginal effects on plantphysiology. These novel crop varieties could, in combinationwith fortification and dietary interventions eradicate MNM,alleviating a great global burden.

AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectualcontribution to the work, and approved it for publication.

FUNDING

SS is indebted to the Agency for Innovation by Science andTechnology in Flanders (IWT) for a predoctoral fellowship.DVDS acknowledges support from Ghent University (BijzonderOnderzoeksfonds, BOF2009/G0A/004), and the ResearchFoundation—Flanders (FWO, project 3G012609).

ACKNOWLEDGMENTS

The authors thank Jolien De Lepeleire for the helpful suggestionsand critical comments on the manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found onlineat: https://www.frontiersin.org/articles/10.3389/fpls.2018.00443/full#supplementary-material

TABLE S1 | An overview of the abbreviations.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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